Use of microphthalmia for diagnosis, prognosis and/or treatment of melanoma

ABSTRACT

Microphthalmia (Mi) while present in melanocytes, a cells and osteoclast, is not normally present in other cells. We have found that Mi is present in the nucleus of melanoma cells. Melanoma can be diagnosed by contacting a malignant cell with a probe for Mi. If the probe identities Mi in the nucleus of the cell, the cell is a melanoma.

GOVERNMENT SUPPORT

This invention was made with Government Support under Grant No. AR43369awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention is directed to methods for diagnosis and/orprognosis of melanoma in individuals using microphthalmia as a marker.

BACKGROUND OF THE INVENTION

Melanoma has been on the rise for decades. It is presently the seventhmost common cancer in the United States. It is currently estimated thatby the year 2000 the lifetime risk of developing skin melanoma inAmericans will be 1 in 75. The annual incidence of human melanomaworldwide is increasing at the rate of approximately 5% per year (16,17). Due to its propensity to metastasize early, coupled to the commonfeature of late recurrence, relapses from melanoma represent animportant and often life threatening clinical condition. The cancerstarts in the skin, but frequently spreads. It can spread locally orthroughout the body. Tissues with melanomas can include lymph glands,liver, bones, brain, lung, adrenal glands, the spinal cord, andvertebrae. Although, once melanoma has spread beyond the original skinsite it is currently considered incurable, there are treatmentmodalities that can prolong an individual's survival.

Metastatic diseases of unknown origin are fairly common. Melanomaresides among the tumor types more commonly associated with metastaseslacking an obvious primary tumor site (18-22). It has proven difficultto determine if such metastatic tissue is melanoma. One of the problemsis that when a melanoma is not found on the skin, its diagnosis isproblematic. Currently, there are two markers typically used to diagnosemelanoma. These markers have problems because the first marker, S100,while sensitive and present in about 80% of melanoma, is also widelypresent in non-melanoma tumors [Kahn, H. J., et al., American J. ofClin. Pathol. 79:341-471 (1983). Thus, it also stains a significantnumber of nonmelanoma malignancies. The second marker, HMB-45, whilevery specific for melanoma, only detects about 50% of melanomas [AppliedImmunolohistochemisty 4:73-95 (1996)]. Other estimate for HMB-45 haveranged as low as 5% and it has been suggested that it stains variably ina technique-dependent fashion (23, 27, 29, 30, 32-35). Thus, there is aneed for other markers that will specifically detect melanoma.

Determining the origin of a metastatic tissue arising from a melanoma isextremely difficult. For example, a skin melanoma can be removed, yetcome back years later at a different site. Conversely, the fact thatsomeone had a melanoma removed 20 years ago, does not mean that ametastatic disease of unknown origin would necessarily be a melanomabecause that individual could have developed a different cancer. Thus,the ability to determine the origin of a metastatic disease is veryimportant because it can affect the diagnosis and/or the type oftreatment regime prescribed. It would be extremely important if a betterand more accurate means for diagnosing melanoma was available. It wouldalso be important having a better means to determine prognosis.

Another problem with melanomas involves the treatment thereof. It isimportant to be able to selectively treat the malignant tissue and notthe surrounding normal tissues. Side effects are frequently experiencedfrom current treatments because some normal tissue is also harmed. Meansfor improving the selectivity are desired.

SUMMARY OF THE INVENTION

We have now discovered an improved method that can be used for diagnosisof melanoma. We have discovered that the transcription factormicrophthalmia (Mi) is an excellent marker that when present in amalignant tissue is diagnostic of melanoma. Mi is normally present inmelanocytes, mast cells, and osteoclasts. However, it is typically notpresent in other cells. Thus, by obtaining a biological specimen,wherein the specimen is preferably a malignant tissue and measuring forthe presence of Mi, by looking at the protein or transcript for Mi, onehas a simple method for the diagnosis of melanoma, wherein the presenceof Mi is indicative of melanoma.

Additionally, by looking at the quantity of Mi, present in the celland/or its state, i.e., activated vs. non-activated, one can use Miprognostically.

Finally, one can take advantage of Mi's correlation with melanoma for amethod of treatment. For example, by selectively targeting Mi one is ineffect selectively targeting melanoma.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show Kit-induced Mi phosphorylation. In FIG. 1A, 501melcells were exposed to SI (20 ng/ml) or TPA (10 ng/ml) for the indicatedtimes and total protein blots were probed for Mi (top), phosphotyrosine(center), and ERK-1/ERK-2 (anti-MAPK, bottom). Bands that reacted withanti-Kit and anti-ERK antibodies are indicated. MAPK activationcorrelates with the Mi mobility shift. FIG. 1B shows reversal of the Mimobility shift with phosphatase. Mi immunoprecipitates fromSI-stimulated cells were treated with increasing amounts of phosphatasefollowed by Western blotting. Phosphatase inhibitors (final lane)prevent the reversal of the mobility shift.

FIGS. 2A and 2B show serine phosphorylation of Mi is prevented by MEKinhibition. FIG. 2A shows that phosphoamino acid analysis of Mi upperband reveals only phosphoserine residues. Mi from ³²P-labeled cells wasimmunoprecipated and the upper band was subjected to acid hydrolysisfollowed by phosphoamino acid analysis by thin layer chromatography.Migration of phosphoamino acid standards are shown at the right. FIG. 2Bshows the effect of MEK inhibition on the Kit- and TPA-stimulated Miphosphorylation. 501mel cells were stimulated by SI or TPA in thepresence of the indicated amounts of the MEK inhibitor, followed bydetection with antibodies against Mi (top) or phospho-MAPK (bottom).

FIGS. 3A-3E show Mi phosphorylation at S73 by MAPK. FIG. 3A, Lysatesfrom +/−SI stimulated melanoma cells were immunoprecipitated (anti-ERK-2or control) and tested in IVK using N-terminal (N) or C-terminal (C)recombinant Mi substrates or MBP. FIG. 3B, Mutations in Mi were testedas IVK substrates from +/−SI cell extracts immunoprecipitated withanti-ERK and identified Ser73 as phosphoacceptor. FIG. 3C, 2D trypticmaps from ³²P labeled endogenous cellular Mi lower and upper bandsrevealed a distinct Kit-dependent spot (hatched circle) which comigrateswith ERK/IVK phosphorylated Mi. “c” and “e” refer to chromatographic andelectrophoretic migration. FIG. 3D, HPLC fractionation of in vivo³²P-labeled Mi tryptic digests from upper band (open diamonds) showscoelution with ERK/IVK recombinant Mi (filled triangles). Y axesindicate beta counts. The secondary peak likely results from oxidativepeptide bond cleavage from performic acid (N. Ahn et al., Curr. Opin.Cell Biol. 4, 992-999 (1992)). FIG. 3E shows wild type or S73A mutant Miwere transfected into COS-7 cells followed by TPA stimulation asindicated. S73A fails to undergo mobility shift.

FIGS. 4A and 4B show MAPK phosphorylation enhances Mi-dependenttransactivation. FIG. 4A, wild type or S73A mutant Mi wereco-transfected with minimal or tyrosinase promoter-driven luciferasereporters into BHK cells. Constitutively active Raf and wild type MEKwere included as indicated to activate the MAP kinase pathway. Mean foldactivation (normalized to 100% for activity of wild type Mi in theabsence of Raf/MEK, column 8) from three independent experiments isshown with standard error bars. Raf/MEK potentiates transactivation bywild type but not S73A Mi. FIG. 4B, Model for signal transduction fromKit to Mi. Tissue-specific factors are shown in grey boxes.

FIGS. 5A-C show identification of human Mi. FIG. 5A shows RT-PCR of Miin neuroblastoma and melanoma cell lines. Mi product migrates as adoublet at 432 bp and 448 bp (18-bp alternative splice (11)).Neuroblastomas were PCR negative while melanomas were positive. FIG. 5Bshows Western blot showing 52 and 56 Kd isoforms of Mi protein (12)(absent in NIH3T3). Steel factor (c-Kit ligand) stimulation of the cellline 501-mel causes phosphorylation of the 52 Kd species as described(12). FIG. 5C is nuclear immunostaining of 501-mel cells with D5 Miantibody. Control lacked primary antibody. 10× and 60× refer tomagnification.

FIG. 6 shows immunohistochemical staining of Normal skin withHematoxylin and Eosin (H&E) and Microphthalmia (Mi) antibody shows Minuclear staining of melanocytes at the epidermal/dermal border. BenignNevus, Dysplastic Nevus and Melanoma In situ show Mi staining in themelanocytic component of these lesions. Arrows indicate areas of Mipositive staining. Asterix indicates endogenous melanin pigment.

FIG. 7 shows immunohistochemistry of melanomas. Conventional melanomarepresents a primary melanoma (epidermis and dermis shown at 40× power)stained with Hematoxylin and Eosin (H&E), Microphthalmia (Mi), HMB-45and S-100. Amelanotic (unpigmented) melanoma is shown at 60×. Metastaticrefers to melanoma within a lymph node (60×). In Transit refers toprimary melanoma with deep dermal invasion without contiguous epidermalinvolvement, at low (10×) and high power (60×).

FIG. 8 shows Mi staining of S-100 negative and HMB-45 negativemelanomas. HMB-45 neg refers to a conventional melanoma that wasnegative for HMB-45 but positive for Mi (arrow). HMB-45 insitu-selective is a case where HMB-45 was positive within the in situcomponent (small arrow), but not the invasive melanoma component(“invasive”) while Mi stained both in situ (small arrow) and invasivecomponents. S-100 neg refers to case where Mi was positive, but HMB-45and S-100 were negative. Deep staining refers to a case where anti-Miidentified melanoma nests deep in the dermis (circled), not easilyappreciated by H&E or HMB-45.

DETAILED DESCRIPTION OF THE INVENTION

Mi is a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) transcriptionfactor implicated in pigmentation, mast cells and bone development. Miis essential to the development and survival of melanocytes.

The gene encoding mouse Mi was cloned in 1993 and found to encode aMyc-related b-HLH-ZIP protein. [Hughes, J. J. et al., J. Biol. Chem.268:20687-20690 (1993); Hodgkinson, C. A. et al., Cell 74: 395-404(1993)] Biochemical studies demonstrated a DNA binding specificity forconsensus sequence CA(C/T)(G/A)TG and its capacity to heterodimerize invitro with three structurally related b-HLH-ZIP factors, TFEB, TFE3, andTFEC, but not Myc/Max, USF or other b-HLH-ZIP proteins. [Hemesath, T.J., Genes & Dev. 8: 2770-2780 (1984); Carr, C. S., et al., Mol. CellBiol., 10: 4384-4388 (1990); Beckman, H., Genes & Dev., 4: 167-179(1990); Roman, C. A., Mol. Cell Biol. 12(2): 817-827 (1992); Zhao, G.Z., et al., Mol. Cell. Biol., 13: 4505-4512 (1993); Yasumoto, K., etal., Biochimica et. Biophysica Acta, 1353: 23-31 (1997); Blackwood, E.M., Science, 251: 1211-1217 (1991)] Mi/Mi mutant mice display defectiveeye development (related to pigment cell abnormalities), complete lackof skin melanocytes, deafness related to absent of pigment cells in theinner ear (stria vascularis)), severe defects in mast cells, andosteoporosis. Mutations in human Mi have been detected in the autosomaldominant hereditary deafness and pigmentation condition, Waardenburgsyndrome, type 2A [Tassabehji, et al., Nature Genet., 8:251-255 (1994);Hughes, A. E. et al., Nature Genet., 7:509-512 (1994)] (a conditioncharacterized by a white forelock and deafness). The Mi gene (4) encodesa transcription factor (5) which regulates expression of thepigmentation enzymes tyrosinase, TRP1, and TRP2 (5-7). Recent studieshave demonstrated that Melanocyte Stimulating Hormone (α-MSH)upregulates pigmentation through stimulation of Mi expression (8, 9).

While Mi may regulate pigmentation, the complete absence of melanocytesin Mi-deficient mice suggests that Mi is essential for melanocytedevelopment or postnatal survival, or both. One instructive mousemutant, mi^(vit) displays nearly normal melanocyte development, butaccelerated age-dependent melanocyte death over the first months of life(10). This death is attributable to a mutation within thehelix-loop-helix motif of mi (5, 11) and suggests a vital role for Mi inpost-natal survival of melanocytes. One potential clue to Mi's survivalrole comes from evidence that the Steel/Kit cytokine pathway (whosedeficiency produces identical absence of melanocytes) regulates MAPkinase-mediated phosphorylation of Mi (12). This producestranscriptional super-activation by Mi protein through selectiverecruitment of CBP/p300 (13), a family of transcriptional coactivatorsfor Mi (14, 15).

Various abnormalities in Mi have been connected to pigmentation deafnessand osteoporosis as stated above. However, its correlation with melanomacells has heretofore been unknown. For example, Mi is involved in asignaling pathway linked to Kit signaling. However, the presence of Kitdoes not correlate with melanoma, despite the fact that Kit is anoncogene. We have now discovered that there is a high correlationbetween the presence of Mi in a malignant cell and that cell being amelanoma. We have been able to determine that Mi is specific formelanoma. For example, we have looked at numerous malignant tissuesincluding many brain tissues and found that Mi was not present. Thenegative Mi staining tumors include basal cell carcinoma, squamous cellcarcinoma, atypical fibroxanthoma, granular cell tumor, Schwannoma andneurofibroma. Thus, by looking for the presence of Mi one is able todetermine the origin of the cell and use such information to determinethe course of treatment.

Mi staining in melanomas produces a nuclear pattern which has sometheoretical advantages over cytoplasmic immunostains. It may bedifficult to distinguish background staining from positivity forcytoplasmic antibodies, especially with weak signal. Furthermore,cellular architecture is not obscured with nuclear staining which aidsin the preservation of the tissue structure being examined. Forpigmented lesions it may be difficult to distinguish cytoplasmic stainsfrom pigmentation, although such lesions are less likely to requirespecial stains.

In one series of experiments, Mi was expressed in 8/8 histologicamelanotic melanomas. Based on its recognition of the M box promoterelement (5), Mi is thought to regulate transcription of the pigmentationenzymes tyrosinase, TRP1 and TRP2 (5-7). Its persistent expression inthe amelanotic melanomas examined here suggests that factors downstreamof Mi may downregulate pigmentation. One such mechanism is theproteolytic degradation of tyrosinase, recently described for humanmelanoma cells (25). These findings suggest that downregulation ofpigmentation is beneficial to melanoma cells and that rather than lossof Mi itself, mechanisms downstream of Mi may more commonly occur. Ofnote, nondetection of Mi expression at the RNA level has been observedin a murine amelanotic melanoma cell line (15).

Mi is a sensitive marker for this clinical entity, which can represent adiagnostic challenge. Due to its propensity for vertical growth,malignant melanoma may metastasize at an early stage, even before aprimary cutaneous lesion is identified (as occurs in 5-14% of cases(18-22)). Moreover since a significant fraction of metastatic melanomasare amelanotic, such lesions may be difficult to classify on simplemorphologic grounds, certainly to the non-specialist, and couldrepresent a variety of undifferentiated or poorly differentiated tumorssuch as epithelial tumors, sarcomas, lymphoid neoplasms or germ celltumors (38). Combined detection of S-100 and Keratin may help rule in orout the possibility a melanoma. S-100 is sensitive for melanoma, butcommonly stains other tumors in this differential including breastadenocarcinomas, lung carcinomas, teratomas, neurogenic tumors, andothers (23, 29) (39-42), whereas Keratin expression is atypical inmelanomas (43).

2 of the breast cancer specimens produced cytoplasmic Mi staining. Mi isexpressed in osteoclasts (37), and many breast cancers express genesinvolved in bone resorption such as PTH-rp, cathepsin K, IL-6, IL-1,TGF, and collagenases (44, 45). Mi may upregulate osteoclast-like genessuch as cathepsin K, a resorption factor recently detected in breasttumor lines (46). As such, Mi expression may play a role in bonemetastasis of breast cancer, perhaps even predicting osteotrophism.Accordingly, targeting Mi expression may also be useful in treatingand/or diagnosing breast cancer.

Mi was not detected in 9 desmoplastic/neurotropic melanomas.Desmoplastic melanomas account for less than 1% of melanomas and oftenarise in association with lentigo maligna (47). About 20-30% of thesetumors lack an in situ component. Desmoplastic tumors tend to grow as afibrous nodule, frequently track along nerves, and have a distinctclinical behavior compared with other melanomas. HMB-45 is oftennegative in desmoplastic melanomas, but S-100 is usually positive. Whilethis tumor is classified as a melanoma, there is some debate as to theorigin and true biology of the spindle cells (48-50), and lack of Mi isbelieved to be notable.

Metastatic melanoma tissue can be present throughout the body and suchlocations typically include lymph glands, liver, bones, brain, lung,adrenal glands, spinal cord and vertebrae. However, malignant tissuespresent in such sites can be from numerous types of cancers. Thus,obtaining a biological sample and looking for the presence of Mi isimportant in being able to diagnose the tissue as a melanoma. Since Miis normally present in melanocytes, mast cells and osteoclasts, thebiological specimen preferably does not include those cells.

Standard detection techniques well known in the art for detectingproteins, RNA, DNA, and peptides can readily be applied to detect Mi orits transcript.

Such techniques may include detection with nucleotide probes or maycomprise detection of the protein by, for example, antibodies or theirequivalent. Preferably, the nucleotide probes may be any that willselectively hybridize to Mi. For example, it will hybridize to Mitranscript more strongly than to other naturally occurring transcriptionfactor sequences. Types of probes include cDNA, riboprobes, syntheticoligonucleotides and genomic probe. The type of probe used willgenerally be dictated by the particular situation, such as riboprobesfor in situ hybridization, and cDNA for Northern blotting, for example.Detection of the Mi encoding gene, per se, will be useful in screeningfor conditions associated with enhanced expression. Other forms ofassays to detect targets more readily associated with levels ofexpression—transcripts and other expression products will generally beuseful as well. The probes may be as short as is required todifferentially recognize Mi mRNA transcripts, and may be as short as,for example, 15 bases, more preferably it is at least 17 bases. Stillmore preferably the Mi probe is at least 20 bases.

A probe may also be reverse-engineered by one skilled in the art fromthe amino acid sequence of Mi. However use of such probes may belimited, as it will be appreciated that any one given reverse-engineeredsequence will not necessarily hybridize well, or at all with any givencomplementary sequence reverse-engineered from the same peptide, owingto the degeneracy of the genetic code. This is a factor common in thecalculations of those skilled in the art, and the degeneracy of anygiven sequence is frequently so broad as to yield a large number ofprobes for any one sequence.

The form of labeling of the probes may be any that is appropriate, suchas the use of radioisotopes, for example, ³²P and ³⁵S. Labeling withradioisotopes may be achieved, whether the probe is synthesizedchemically or biologically, by the use of suitably labeled bases. Otherforms of labeling may include enzyme or antibody labeling such as ischaracteristic of ELISA.

Detection of RNA transcripts may be achieved by Northern blotting, forexample, wherein a preparation of RNA is run on a denaturing agarosegel, and transferred to a suitable support, such as activated cellulose,nitrocellulose or glass or nylon membranes. Radiolabelled cDNA or RNA isthen hybridized to the preparation, washed and analyzed byautoradiography.

In situ hybridization visualization may also be employed, wherein aradioactively labeled antisense cRNA probe is hybridized with a thinsection of a biopsy sample, washed, cleaved with RNase and exposed to asensitive emulsion for autoradiography. The samples may be stained withhaematoxylon to demonstrate the histological composition of the sample,and dark field imaging with a suitable light filter shows up thedeveloped emulsion. Non-radioactive labels such as digoxigenin may alsobe used.

Immunohistochemistry is preferably used to detect expression of human Miin a biopsy sample. A suitable antibody is brought into contact with,for example, a thin layer of cells, washed, and then contacted with asecond, labeled antibody. Labeling may be by enzyme, such as peroxidase,avidin or by radiolabelling. Chromogenic labels are generallypreferable, as they can be detected under a microscope. Mi is a nuclearprotein and provides a good staining pattern.

More generally preferred is to detect the protein by immunoassay, forexample by ELISA or RIA, which can be extremely rapid. Thus, it isgenerally preferred to use antibodies, or antibody equivalents, todetect Mi.

It may not be necessary to label the substrate, provided that theproduct of the enzymatic process is detectable and characteristic in itsown right (such as hydrogen peroxide for example). However, if it isnecessary to label the substrate, then this may also comprise enzymelabeling, labeling with radioisotopes, antibody labeling, fluorescentmarker labeling or any other suitable form which will be readilyapparent to those skilled in the art.

Antibodies may be prepared as described below, and used in any suitablemanner to detect expression of Mi. Antibody-based techniques includeELISA (enzyme linked immunosorbent assay) and RIA (radioimmunoassay).Any conventional procedures may be employed for such immunoassays. Theprocedures may suitably be conducted such that: a Mi standard is labeledwith a radioisotope such as ¹²⁵I or ³⁵S, or an assayable enzyme, such ashorseradish peroxidase or alkaline phosphatase and, together with theunlabelled sample, is brought into contact with the correspondingantibody, whereon a second antibody is used to bind the first andradioactivity or the immobilized enzyme assayed (competitive assay);alternatively, Mi in the sample is allowed to react with thecorresponding immobilized antibody, radioisotope- or enzyme-labeledanti-Mi antibody is allowed to react with the system and radioactivityor the enzyme assayed (ELISA-sandwich assay). Other conventional methodsmay also be employed as suitable.

For example using a monoclonal antibody to Mi resulted in strong nuclearstaining within melanocytes, nevi, dysplastic nevi, melanoma in situ,and 100% of 76 consecutively acquisitioned melanomas, includingamelanotic and metastatic tumors. In side by side comparisons Mi stainedtumors which were negative for HMB-45 or S100. Among nonmelanoma tumors,Mi stained cytoplasms in two of 81 cases, and no cases exhibited nuclearstaining. Thus, Mi is a sensitive and specific marker for melanoma.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting antigen withimmobilized antibody and, without washing, contacting the mixture withlabeled antibody. The “two-step” assay involves washing beforecontacting the mixture with labeled antibody. Other conventional methodsmay also be employed as suitable.

Enzymatic and radio-labeling of Mi and/or the antibodies may be effectedby conventional means. Such means will generally include covalentlinking of the enzyme to the antigen or the antibody in question, suchas by glutaraldehyde, specifically so as not to adversely affect theactivity of the enzyme, by which is meant that the enzyme must still becapable of interacting with its substrate, although it is not necessaryfor all of the enzyme to be active, provided that enough remains activeto permit the assay to be effected. Indeed, some techniques for bindingenzyme are non-specific (such as using formaldehyde), and will onlyyield a proportion of active enzyme.

It is usually desirable to immobilize one component of the assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed withoutlaborious and time-consuming labor. It is possible for a second phase tobe immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but ifsolid-phase enzyme is required, then this is generally best achieved bybinding to antibody and affixing the antibody to a support, models andsystems for which are well-known in the art. Simple polyethylene mayprovide a suitable support.

Enzymes employable for labeling are not particularly limited, but may beselected from the members of the oxidase group, for example. Thesecatalyze production of hydrogen peroxide by reaction with theirsubstrates, and glucose oxidase is often used for its good stability,ease of availability and cheapness, as well as the ready availability ofits substrate (glucose). Activity of the oxidase may be assayed bymeasuring the concentration of hydrogen peroxide formed after reactionof the enzyme-labeled antibody with the substrate under controlledconditions well-known in the art.

Other techniques may be used to detect Mi according to preference. Onesuch technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci.76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGEgel before being transferred to a solid support, such as anitrocellulose filter. Anti-Mi antibodies (unlabelled) are then broughtinto contact with the support and assayed by a secondary immunologicalreagent, such as labeled protein A or anti-immunoglobulin (suitablelabels including ¹²⁵I, horseradish peroxidase and alkaline phosphatase).

Samples for diagnostic purposes may be obtained from any number ofsources. A sample obtained directly from the tumor, such as the stromaor cytosol, may be used to determine the origin of the tumor. It mayalso be appropriate to obtain a sample from other biological specimens,where Mi is present. Such diagnosis may be of particular importance inmonitoring progress of a patient, such as after surgery to remove atumor. If a reference reading is taken after the operation, then anothertaken at regular intervals, any rise could be indicative of a relapse,or possibly a more severe metastasis. Preferably, the sample is from thetumor itself.

Mi binds E box-type enhancer elements and may heterodimerize with therelated family members TFEB, TFEC and TFE3 (Hemesath, T. J., et al.,Genes Dev. 8, 2770-80 (1994)). Mutations in c-Kit or its ligand SI (stemcell factor, mast cell growth factor) similarly result in animalslacking melanocytes and functional mast cells, along with defects inhematopoiesis and germ cell development (Russell, E., Adv. Genet. 20,357-459 (1979).

Witte, O. Steel, Cell 63, 5 (1990)). This striking phenotypic overlaphas led to suggestions that SI, c-Kit, and Mi function in a commongrowth/differentiation pathway (Steingrimsson, E., et al., Nature Genet.8, 256-63 (1994); Dubreuil, P., et al., Proc. Natn. Acad. Sci. U.S.A.88, 2341-2345 (1991)). Germline mutations at loci encoding thetranscription factor Mi, the cytokine receptor c-Kit, and its ligandSteel factor (SI) result in strikingly similar defects in mast cell andmelanocyte development (Moore, K. J., Trends Genet. 11, 442-8 (1995);Russell, E., Adv. Genet. 20, 357-459 (1979); Witte, O. Steel, Cell 63, 5(1990).

We found a biochemical link between Kit signaling and the activity ofMi. Stimulation of melanoma cells with SI results in activation of MAPkinase, which in turn phosphorylates Mi at a consensus target serine.This phosphorylation upregulates Mi transactivation of the tyrosinasepigmentation gene promoter. In addition to modulating pigmentproduction, such signaling may regulate the expression of genesessential for melanocyte survival and development. The pathwayrepresents a novel use of the general MAP kinase machinery to transducea signal between a tissue-specific receptor at the cell surface and atissue-specific transcription factor in the nucleus.

The antibodies may be raised against either a peptide of Mi or the wholemolecule. Such a peptide may be presented together with a carrierprotein, such as an KLH, to an animal system or, if it is long enough,say 25 amino acid residues, without a carrier. Preferred peptidesinclude regions unique to Mi.

Polyclonal antibodies generated by the above technique may be useddirect, or suitable antibody producing cells may be isolated from theanimal and used to form a hybridoma by known means (Kohler and Milstein,Nature 256:795. (1975)). Selection of an appropriate hybridoma will alsobe apparent to those skilled in the art, and the resulting antibody maybe used in a suitable assay to identify Mi.

This invention also provides a convenient kit for detecting human Milevels. This kit includes a probe for Mi such as antibodies or antibodyfragments which selectively bind human Mi or a set of DNAoligonucleotide primers that allow synthesis of cDNA encoding human Mi.Preferably, the primers comprise at least 10 nucleotides, morepreferably at least about 20 nucleotides, and hybridizes under stringentconditions to a DNA fragment having the human Mi sequence nucleotide.The kit will contain instructions indicating how the probe can be useddiagnostically or prognostically. As herein used, the term “stringentconditions” means hybridization will occur only if there is at least 95%and preferably at least 97% homology between the sequences.

Homology is measured by means well known in the art. For example %homology can be determined by any standard algorithm used to comparehomologies. These include, but are not limited to BLAST 2.0 such asBLAST 2.0.4 and i. 2.0.5 available from the NIH (Altschul, S. F., et al.Nucleic Acids Res. 25: 3389-3402 (1997)) and DNASIS (Hitachi SoftwareEngineering America, Ltd.). These programs should preferably be set toan automatic setting such as the standard default setting for homologycomparisons. As explained by the NIH, the scoring of gapped resultstends to be more biologically meaningful than ungapped results.

One can also take advantage of Mi's correlation with melanoma to treatmelanoma. Thus, one can screen for and select compounds, preferablysmall molecules that selectively react with Mi. These compounds can thenbe used to provide selective targeting of the melanoma. For example, thesmall molecule could be cytotoxic. In another embodiment, the compound,e.g. a cytotoxic compound such as ricin could bind to Mi and beactivated so that the molecule serves as a target or catalyst for asecond compound that it used to treat a melanoma.

All references cited above or below are herein incorporated byreference.

The following Examples serve to illustrate the present invention, andare not intended to limit the invention in any manner.

EXAMPLES Immunoprecipitation (IP) and Western Blot Analysis

The monoclonal antibody D5 was raised against a histidine fusion proteinexpressed from the amino terminal Taq-Sac fragment of human MITF cDNA(Tachibana, M., et al., Hu. Mol. Genet. 3, 553-7 (1994)) and produces aspecific gel mobility supershif with Mi, but not with the relatedproteins TFEB, TFEC, and TFE3 (not shown). 501mel cells (gift of Dr.Ruth Halaban, Yale University) were maintained in F10 medium plus 10%fetal calf serum (FCS). Cells were stimulated with 20 ng/ml recombinanthuman SI (R&D Systems) or 10 ng/ml TPA for eight minutes at 37° C.unless otherwise indicated. Cells were lysed in 50 mM Tris pH 7.6, 150mM NaCl, 10% Triton-X 100 plus protease and phosphatase inhibitors.Samples were solubilized in SDS sample buffer plus 0.5%2-mercaptoethanol and boiled for 5 minutes. Following SDS/PAGE andtransfer to nitrocellulose, blots were blocked in 5% milk/0.05% Tween-20in Tris-buffered saline prior to antibody incubation. Proteins weredetected with peroxidase-conjugated second-step antibody (Cappel) andchemiluminescence reagents (Amersham).

Phosphatase Treatment.

Immunoprecipitated Mi was washed three times with lysis buffer, twicewith Buffer A (100 mM KCl, 20 mM Hepes pH 7.4, 0.2 mM EDTA, 2 mM DTT,plus protease inhibitors), and resuspended in 40 μl Buffer A. Controldigests contained phosphatase inhibitors sodium vanadate (1 mM), sodiumpyrophosphate (20 mM), and sodium fluoride (10 mM). Potato acidphosphatase (Boehringer Mannheim) was added to IPs for 15 minutes at30°. The reaction was stopped with phosphatase inhibitors and analyzedby Western blot.

Phosphoamino Acid Analysis, Tryptic Mapping, HPLC Fractionation.

501mel cells were starved for 30 minutes in serum-free, phosphate-freeRPMI medium, then labeled for 3 hours using 1 mCi/ml ³²P inorganicphosphate. Cells were stimulated with SI or TPA and solubilized in IPbuffer. Mi proteins were immunoprecipitated overnight at 4° C.,electrophoresed and transferred to nitrocellulose. Bands were cut outand digested 20 hours at 37° with 25 μg TPCK-treated trypsin (SIGMA).Phosphoamino acid analysis and phosphopeptide mapping were carried outas described (Boyle, W., et al., Methods Enz. 210, 110-149 (1991)) usingpH 8.9 ammonium carbonate. HPLC fractionation utilized a 25 cm C18reverse phase column (VYDAC®) and an acetylnitrile gradient (0-70% in0.1% trifluoracetic acid) at a flow rate of 0.2 ml/minute. Fractionswere assayed by Cerenkov counting.

In Vitro Kinase Assay.

Cells were activated and lysed as described and 4 μl of anti-ERK-2antiserum (Santa Cruz) plus 20 μl of Protein A agarose beads were addedto the lysate and mixed at 4° overnight. Beads were washed three timeswith lysis buffer and once with IVK buffer (50 mM Hepes pH 7.6, 2 mMsodium vanadate, 10 mM magnesium chloride, 1 mM PMSF, 2 mM DTT and 50 μMATP). For each reaction, 40 μl IVK buffer, 1 μl γ³²p. ATP, andphosphoacceptor protein were added. Myeline basic protein (5 μg) or Mihistidine fusion proteins Taq-Taq, Taq-Sac, or Bam-Bam (Tachibana, M.,et al., Hu. Mol. Genet. 3, 553-7 (1994)) (4 μl at 0.065 ²⁸⁰OD) wereadded as substrates and incubated at 30° C. for 30 minutes. Reactionswere stopped by addition of 2×SDS sample buffer and analyzed by Westernblot and autoradiography.

Luciferase Assay.

The human tyrosinase promoter reporter encompasses nucleotides −300 to+80 (Bentley, N. J., et al., Mol. cell. Biol. 14, 7996-8006 (1994)) inthe pGL2Basic luciferase reporter (Promega). Wild type Mi and the S73Amutant were cloned into the pEF-BOS expression vector (Mizushima, S., etal., Nucl. Acids Res. 18, 5322 (1990)). The plasmid encodingconstitutively active Raf was the 24G deletion mutant (Stanton, V., etal., Mol. cell. Biol. 9, 639-647 (1989)), a gift from Dr. GeoffreyCooper (Dana-Farber Cancer Institute, Boston, Mass.). Wild type MEKplasmid was a gift from Dr. Len Zon (Children's Hospital, Boston,Mass.). Transfections were performed by adding plasmid DNA (10 μg totalfor 6 cm plate) to 300 μl DMEM, mixing 1:1 with a 5% lipofectamine/DMEMsolution, and incubating at room temperature for one hour. BHK cellsmaintained in DMEM/10% FCS were washed twice with serum-free DMEM priorto transfection. DNA/lipofectamine was added to 2 ml DMEM on a 6 cmplate. Cells were incubated overnight at 37° and fed the next morning.Assays were harvested 8 hours later and analyzed with a Moonlight 2010Luminometer using reagents and recommendations of the manufacturer(Analytical Luminescence Laboratory). Luciferase data were normalized toμ-galactosidase activity in all samples.

Immunohistochemistry: Mi antibodies were generated against theN-terminus Taq-Sac fragment of human Mi expressed as His-fusion andshown not to cross react with other b-HILH-ZIP factors by DNA mobilityshift assay (data not shown).

Cells are grown on glass chamber slides (Fisher Scientific, Pittsburgh,Pa.) and will be fixed with 3% formaldehyde in PBS for 30 minutes andwashed followed by 10 minutes in 1% Triton X-100. After another PBSwash, slides will be incubated w/3% H₂O₂ to remove endogenousperoxidase. The anti-Mi monoclonal antibody 1:10-1:40 or anti-TFE3monoclonal antibody (PharMingen, San Diego, Calif.) 1:250-1:500 will beadded for 1 hour. The VECTASTAIN® Elite kit (Vector Laboratories,Burlingame, Calif.) is then used for immunohistochemical stainingaccording to manufacturer's instructions. The diaminobenzidine (DAB)reagent (Vecta Laboratories) is applied for 2-4 minutes and the slidesare analyzed under light microscopy.

Immunoprecipitation/Western blotting: Immunoblots of melanoma cells aregenerally preceded by immunoprecipitation which concentrates the antigenand permits efficient on-plate cell lysis in 1% Triton X-100 detergentwith 150 mM NaCl, Tris PH 7.6, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 200μg/ml trypsin inhibitor, 500 μg/ml antipain, 10 mM sodium fluoride, 1 mMsodium vanadate, 2 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM sodiumpyrophosphate, 10 μg/ml pepstatin. The soluble fraction is incubated for1-2 hours on ice with appropriate antibodies and washed protein Aagarose beads (Gibco-BRL, Gaithersburg, Md.) are added. The mixture isrotated at 4° C. for a minimum of 12 hours. Beads are then washed 3times with PBS, resuspended in 2% SDS/1% glycerol, boiled for 5 minutes,and eluted proteins are resolved on 8% SDS/polyacrylamide gels (4%SDS/polyacrylamide stacking gel) run at 200 volts for 6-8 hours.Proteins are transferred to nitrocellulose with methanol/glycineelectrotransfer (BioRad, Hercules, Calif.). The membrane is blocked in5% milk for 1 hour at room temperature or overnight at 4° C. Afterwashing in 10 mM Tris pH 7.6, 150 mM NaCl, 0.5% Tween (TBST), 1:40dilution of the Mi antibody or 1:500 dilution of TFE3 antibody or alphatubulin 1:500 dilution (Sigma, St. Louis, Mo.) is added for 1 hour atroom temperature. After washing, goat-anti-mouse horseradish peroxidaseconjugated antibody (Cappel, West Chester, Pa.) is added for 40 minutes.After washing, the enhanced chemiluminescence reaction is performed(Amersham, Arlington Heights, Ill.).

PCR and Southern Analysis of Genomic DNA. Genomic DNA can be extractedform lenanocytes, or mast cells using the PUREGENE™ kit (Gentra Systems,Plymouth, Minn.) according to manufacturer's instructions. The genomicPCR reactions employ 50 ng of purified genomic DNA under the followingconditions: 94° C. for 2 minutes, 57° C. for 1 minute and 72° C. for 2minutes except for exon 9 where an annealing temperature of 60° C. for 1minute was used for 30 cycles. For Southern analysis, 10 μg of genomicDNA is digested with each of the following restriction enzymes Hinc II,Xba I, and Bam HI using the manufacturer's instructions (New EnglandBiolabs). The digested DNA is electrophoresed on a 1% agarose/TBE gel.The gel is denatured with 0.25 M HCI followed by 0.5 M. NaOH/1.5 M NaCl,and equilibrated with Tris pH 8.0/1.5 M NaCl. The DNA is transferred tonylon membranes, UV crosslined, and prehybridized for 30 minutes withQuik-Hyb (Strategene, LaJolla, Calif.) at 65OC. ³²P-dCTP radiolabeledfull length Mi cDNA is made using the Strategene prime-it randomlabeling kit. 10⁶ cpm/ml of Quik-Hyb solution is used and hybridizationis performed at 65OC for 2 hours.

RT-PCR/Northern analysis: Total cellular RNA from a malignant tissue andcultured cells are isolated using RNAZOL™ (Tel-Test, Friendswood, Tex.)according to the manufacturer's instructions. A series of primers, forexample, to mouse microphthalmia exons 5, 6, 7, 8 and 9 are synthesized:

5′ exon 5 CCGTCTCTGGAAACTTGATCG; (SEQ ID NO:1) 5′ exon 8GACATGCGGTGGAACAAGGG; (SEQ. ID NO:3) 5′ exon 9 GAGCTGGAGATGCAGGCTAG;(SEQ. ID NO:4) 3′ exon 5 GTTGGGAAGGTTGGCTGGAC; (SEQ. ID NO:5) 3′ exon 6CTGCCTCTCTTTAGCCAATGC; (SEQ. ID NO:6) 3′ exon 7 GGATCATTTGACTTGGGGATCAG;(SEQ. ID NO:7) 3′ exon 8 CTGTACTCTGAGCAGCAGGTG; (SEQ. ID NO:8) 3′ exon 9GCTCTCCGGCATGGTGCCGAGG. (SEQ. ID NO:9)cDNA are made using the Gibco BRL RT-PCR kit (Grand Isle, N.Y.). 1-5 μgof total RNA are used for each reaction along with 30-100 pmol of 3′primer and 200 units of SUPERSCRIPT II™ reverse transcriptase andincubated for 50 min at 42° C., 2-4 μl of the 20 μl cDNA reactionmixture is used for PCR reactions which contained: Taq polymerase(Fisher Scientific), dNTP's (Pharmacia Biotech), and PCR buffer (PerkinElmer) and with incubations at 94° C. for 2 minutes, 57° C. for 1 minuteand 72° C. for 2 minutes for 30 cycles (Annealing temperatures forreactions containing 5′ exon 6 are 67° C.; for 5′ exon 7 was 57° C.; andfor 5′ exon 8 and 5′ exon 9 are 62° C.). 25-50 μl of the 100 μl reactionmixtures are resolved by electrophoresis in non-denaturing 8-0%polyacrylamide gels. Negative controls are performed with water in placeof DNA. The RT-PCR product form 5′ exon 5 and 3′ exon 8 for wild typerat is gel purified and sequenced. The sequence is confirmed from 4independent PCR reactions. Mi expression is examined using Northernanalysis with 20 μg of total RNA loaded on a formaldehyde gel,transferred to nylon membranes, and probed with radiolabeled full lengthmi DNA for 2 hours at 65° C. in QUICK HYB® solution. For loadingcontrol, a radiolabeled 500 bp Xba I/Hind III fragment of the humanglyceraldehyde 3′ phosphate dehydrogenase (GAPCH) is used.

Cell Lines, RT-PCR, and Western blotting: NIH3T3 murine fibroblasts, B16murine melanoma, and human neuroblastoma lines IMR-32 and SK-N-SH weregrown in DMEM with 10% fetal bovine serum (FBS). The human melanoma celllines (gift of Dr. R. Halaban, Yale University) 501-mel (24), MeWo, andYUZAZ6(36) were grown in Ham's F10 media supplemented with 10% FBS.RT-PCR and Western blotting were carried out as described (37). Primersto the mi gene flanking exons 5-8 were:

5′ exon 5 CCCGTCTCTGGAAACTTGATCG (SEQ ID NO:8) and 3′ exon 8CTGTACTCTGAGCAGCAGGTG. (SEQ ID NO:10)

Immunofluorescence and immunohistochemistry: Mi monoclonal antibodies(12, 37) failed to crossreact with other b-HLH-Zip factors byimmunoprecipitation and DNA mobility shift assay ((37) & data notshown). C5 recognizes mouse and human Mi and was used for Westernblotting. D5 recognizes human Mi only and was used for immunostaining.For staining, cells (grown on glass chamber slides (Fisher Scientific,Pittsburgh, Pa.)) were fixed with 3% formaldehyde in phosphate bufferedsaline (PBS) for 30 minutes. D5 antibody (diluted 1:40) was added for 1hour. The VECTASTAIN® Elite kit (Vector Laboratories, Burlingame,Calif.) was used for immunohistochemical staining per manufacturer'sinstructions. The diaminobenzidine reagent (Vector Laboratories) wasapplied for 2-4 minutes. For immunofluorescence, the Cy-3 conjugatedgoat anti-mouse (Jackson Immunological) was used. Nuclei were stainedwith 10 ng/ml DAPI (Sigma). All incubations were followed by threewashes with 0.1% Triton X-100 in PBS.

Histopathology: 80 sequential cases of melanoma were selected from thepathology files of Albany Medical Center. Of these, 4 were excluded dueto unavailability of adequate lesional tissue. Histopathology wasreviewed by two of the authors (RK and MM) to confirm all diagnoses.Immunohistochemical studies were performed utilizing formalin fixedparaffin-embedded tissue. Sections were cut at 4 micrometers, heated at60° C., deparaffinized in xylenes, and hydrated in a graded series ofalcohols. Primary antibodies included rabbit antibody S-100 (Ventana,prediluted), mouse monoclonal antibody HMB-45 (Ventana, prediluted), andmonoclonal antibody D5 to Mi (undiluted). Antigen retrieval wasperformed using microwaving in citrate buffer for Mi antibody. Stainingwas performed with the Ventana ES automated immunohistochemistry systemusing the Ventana DAB Detection Kit (Ventana Medical Systems Inc.)Tissues known to express the antigen of interest were used as positivecontrols whereas removal of the primary antibodies in the test tissueswere used as negative controls. Nuclear staining for Mi was regarded aspositive whereas cytoplasmic staining alone was considered negative(observed in two breast carcinomas, see below). S-100 antibody stainingwas considered positive if cytoplasmic staining was present and, forHMB-45, cytoplasmic staining was considered positive. In addition, 9cases of desmoplastic/neurotropic melanoma and 2 cases of pure spindlecell melanoma (not from the consecutive series) were examined with thesame antibodies. 81 non-melanocytic tumors (detailed below) wereselected to test the specificity of Mi and HMB-45, and selectedmelanocytic and non-melanocytic skin lesions (detailed below) were alsostained for Mi.

Results

Western blot analysis of a human melanoma cell line revealed two Mispecies with relative mobilities of 54 and 60 kd. Activation of c-Kit bySI completely shifted the lower band to the position of the upper band(FIG. 1A). This shift occurred rapidly but transiently; the lower bandreappeared within two hours. A smaller but sustained shift was inducedwhen cells were treated with phorbol ester (TPA), a potent activator ofprotein kinase C. As detected by phosphotyrosine antibodies, treatmentwith SI but not TPA led to the expected phosphorylation of c-Kit (FIG.1A). Both stimuli resulted in the activation of MAP kinase, whichcorrelated temporally with the shift in Mi protein (FIG. 1A). In vitrophosphatase treatment resulted in a discrete, dose-dependent shift ofthe upper form of Mi to the faster-migrating species (FIG. 1B). Thisshift was blocked by phosphatase inhibitors, suggesting that themobility change observed in cell extracts was due to phosphorylation.

In vivo labeling of the Mi proteins with ³²P-orthophosphate indicatedthat both Mi forms were phosphoproteins (see below). Phosphoamino acidanalysis revealed phosphoserine, but no detectable phosphotyrosine orphosphothreonine (FIG. 2A), a finding consistent with the failure ofanti-phosphotyrosine antibodies to detect Mi in total cell extracts (notshown). These results indicated that c-Kit was not responsible fordirectly phosphorylating Mi, and therefore attention was focused ondownstream kinases.

Phosphotyrosine blotting showed that MAP kinase activation correlatedwith Mi phosphorylation in cells (FIG. 1A). One family of MAPKsactivated by the c-Kit signaling cascade includes ERK-1 and ERK-2(Okuda, K., et al., Blood 79, 2880-7 (1992)) which translocate to thenucleus upon activation and phosphorylate a number of transcriptionfactors (Marshall, C., Curr. Opin. Cell 80, 179-15 (1995); Treisman, R.,Curr. Opin. Cell Biol. 8, 205-215 (1996)). The ERKs are activated by adual phosphorylation event carried out by the upstream kinase MEK-1(Ahn, N., et al., Curr. Opin. Cell Biol. 4, 992-999 (1992)). We examinedthe effect of a specific inhibitor of MEK activity, PD98059 (MEKi), onthe phosphorylation of Mi in vivo. As shown in FIG. 2B, MEKi preventedERK phosphorylation and caused a dose-dependent inhibition of the Mimobility shift stimulated by both SI and TPA. MEKi did not affect thetyrosine phosphorylation of c-Kit in response to SI, indicating that thedrug did not grossly perturb signaling (not shown). These resultssuggested that Mi might be a substrate for activated ERK in vivo.

The ability of ERKs to phosphorylate Mi was tested in vitro usingimmunoprecipitated kinase. ERK-2 strongly phosphorylated anamino-terminal fragment of Mi but failed to phosphorylate a largefragment from the carboxy terminus (FIG. 3A). This in vitrophosphorylation was dependent upon prior activation of the cells with SI(FIG. 3A). Jun N-terminal kinase and p38 kinase did not revealdetectable c-Kit-dependent kinase activity on recombinant Mi proteins(not shown). ERK-1 was not significantly expressed in the cells (notshown). Thus c-Kit stimulation generated activated ERK-2 capable ofphosphorylating Mi in vitro.

The amino-terminal region of Mi contains three closely spaced serineresidues that could potentially act as MAPK phosphoacceptor sites (FIG.3B). Mutation of the two upstream serines (S68 and S69) had no effect onin vitro phosphorylation by ERK-2, while mutation of serine 73completely abolished in vitro phosphorylation (FIG. 3B). Mutation ofproline 71, which would contribute to a S73-directed consensus MAPK site(PXSP) resulted in a severe reduction of ERK-2 phosphorylation (FIG.3B).

To determine whether ERK-2 was the kinase responsible for Miphosphorylation in vivo, we carried out two-dimensional phosphopeptidemapping and high pressure liquid chromatography (HPLC) fractionation ofthe trypsin-digested ³²P-labeled Mi doublet. The patterns derived fromboth Mi bands contain peptides representing constitutivephosphorylations (FIG. 3C, compare Lower vs. Upper). However, a uniquephosphotryptc fragment appears in the map of the upper Mi band, whichcorresponds to the c-Kit-dependent phosphorylation (FIG. 3C, Upper). Acomparison of this spot to that generated from in vitro ERK-2phosphorylated recombinant Mi showed identical two-dimensional migration(FIG. 3C, Upper vs. Recombinant). HPLC fractionation of tryptic digestsfrom in vivo or in vitro phosphorylated protein confirmed that theycontain a single major co-eluting phosphotryptic fragment (FIG. 3D). Thesecond labeled peak eluting from both HPLC fractionations likely resultsfrom oxidative peptide bond cleavage resulting from performic acidtreatment (Boyle, W., et al., Methods Enz. 210, 110-149 (1991)).

The site-directed Mi mutant S73A failed to undergo TPA-induced mobilityshift in transfected cells (FIG. 3E) indicating that phosphorylation ofS73 is most likely responsible for the Kit-dependent mobility shift. Thetryptic maps, HPLC fractionation, and in vitro kinase data stronglysuggest that ERK-2 is activated by the c-Kit signaling cascade andsubsequently phosphorylates Mi at S73.

The impact of MAPK phosphorylation of Mi on its ability to transactivatewas tested on a luciferase reporter driven by the tyrosinase promoter, arate limiting enzyme in the pigmentation response (Hearing, V., et al.,J. Biochem, 19, 1141-1147 (1987)). Mi has been shown to transactivatethis promoter through M box enhancer elements (conserved in thepromoters of all known pigment enzyme genes) (Hemesath, T. J., et al.,Genes Dev. 8, 2770-80 (1994); Bentley, N. J., et al., Mol. cell. Biol.14, 7996-8006 (1994); Yasumoto, K., et al., Mol. Cell Biol. 14, 8058-70(1994)). Due to the transient nature of Kit signals (see FIG. 1),constitutively active Raf plus wild type MEK were used to achievesustained activation of the MAP kinase pathway and permit measurableaccumulation of luciferase. Co-transfection of Raf/MEK resulted inup-regulation of tyrosinase reporter activity in the presence of wildtype Mi (FIG. 4). Mutant S73A showed no significant transcriptionalenhancement in response to Raf/MEK despite expression levels comparableto those of wild type Mi (data not shown, see FIG. 3E). Thus MAPKphosphorylation at serine 73 mediates up-regulation of Mitranscriptional activity.

The phosphorylation of Mi in response to c-Kit activation identifies anuclear target that may underlie the well-documented phenotypic overlapbetween mice bearing mutations in SI, Kit and Mi. It is likely that thespecificity of the MAPK pathway between Kit and Mi lies in the highlyrestricted temporal and spatial expression of SI, Kit and Mi in vivo.Mi/Kit signaling illustrates use of general signaling machinery to linkan individual cytokine to a specific transcription factor (FIG. 4B).These results are consistent with a recent report that subcutaneous SIinduces localized pigmentation in humans (Costa, J., et al., J. Exp. Med183, 2681-2686 (1996)). cAMP elevation may also up-regulate thetyrosinase promoter in a manner involving Mi via signals originatingfrom the melanocyte stimulating hormone receptor (Englaro, W., et al.,J. biol. Chem. 270, 24315-24320 (1995); Bertolotto, C., et al., J. CellBiol 134, 747-755 (1996).

Interestingly, mast cells from mi mutant mice underexpress c-Kit (Ebi,Y., et al., Blood 80, 1454-62 (1992)) and Mi may upregulate c-Kitexpression from a binding site in the c-Kit promoter (Tsujimura, T., etal., Blood 88, 1225-33 (1996)). Additionally, Bernstein and colleaguesfound that transfection of the c-Kit related CSF-1 receptor restorescytokine-responsiveness to kit-mutant but not mi-mutant mast cells(Okuda, K., et al., Blood 79, 2880-7 (1992)), consistent with the roleproposed here for Mi as a downstream target of cytokine-initiated MAPKsignals. Moreover, genetic experiments (Paulson, R., et al., NatureGenet. 13, 309-315 (1996); Lorenz, U., et al., J. Exp. Med. 184,111-1126 (1997)) demonstrated that germline deficiency of thephosphatase SHP1 (a negative regulator of Kit signaling) enhancesSI-induced MAP kinase activation and partially rescues the number ofresident mast cells. Kit-stimulated MAPK activation and Miphosphorylation may alter expression of genes controlling cell lineagecommitment, development or survival.

Using reverse transcriptase PCR, bands corresponding to Mi wereidentified in a series of five melanoma cell lines, four human (24, 36)and one murine. A doublet representing the alternative splice of an 18bp segment (4) was observed in each case (FIG. 5A) and verified bysequencing. Two human neuroblastoma cell lines, also neural crestderived tumors, failed to produce Mi-specific PCR products (FIG. 5A).Western blot analysis of 501-mel melanoma cell line extracts (FIG. 5A,lanes 2-5) revealed Mi-specific bands as a doublet migrating at ˜52 and56 kd. Biochemical analyses have confirmed the identity of these Mibands and determined that these isoforms differ in the presence of a MAPkinase-mediated phosphorylation at serine 73 in the upper migratingspecies (12). A fibroblast extract (FIG. 5B, lane 1) lacked the Miprotein bands. In addition, Steel factor (c-Kit ligand) triggered amobility shift from the lower to the upper migrating form as previouslydescribed (FIG. 6B lanes 6 & 7, (12)). Direct staining of melanoma cellsfor Mi revealed nuclear signal by immunofluorescence andimmunohistochemistry (FIG. 5C). Two color fluorescence with DAPIidentifies nuclei in the same samples (FIG. 5C). Thus, Mi is expressedin nuclei of these melanoma cells.

Expression of Mi was next tested by immunohistochemical staining innormal skin, nevi, dysplastic nevi, and melanomas. Paraffin-embeddedtissue samples were stained with Mi and counterstained with hematoxylin.Control samples from each section were separately stained withhematoxylin and eosin (H & E) for comparison. Within normal skin, the Mispecific antibody highlighted nuclear staining within individualmelanocytes (FIG. 6, see arrows). In addition, melanocytes in 9 benignnevi and 4 dysplastic nevi were all positive for Mi (representativecases shown in FIG. 6).

Mi expression was tested in a series of consecutively accessioned humanpathologic melanoma specimens. Of the 76 cases, 19 were melanomas insitu, 50 were conventional melanomas, and 7 were metastatic melanomas.Eight cases were histologically amelanotic. Nine cases had apredominantly spindled cell morphology, eight cases a mixedspindled/epithelioid morphology, and the remainder a predominantlyepithelioid morphology.

Mi expression was positive and nuclear in all 19 melanomas in situ(Table 1, and FIG. 6). Among the conventional invasive melanomas, Mi wasalso positive in all cases, again displaying a nuclear staining pattern(Table 1). For the consecutive series, Mi staining was compared inside-by-side fashion with HMB-45 and S-100. The nuclear staining patternof Mi contrasted the cytoplasmic or more diffuse staining patterns ofS-100 and HMB-45 (FIG. 7). All three stains were positive in themajority of cases (Table 1). However, HMB-45 failed to stain 7 cases andproduced only rare scattered immunopositivity in 2 more. While known tobe less specific for melanoma (31), S100 was positive in all but 5melanoma cases. Mi staining was positive in all 76 tumors with 2 casesdemonstrating focal positivity. FIG. 7 shows representative staining forMi, HMB-45, and S100 in conventional, amelanotic, metastatic, andin-transit melanomas. The nuclear staining pattern of Mi is compared atlow and high powers. The amelanotic tumor happened also to be negativefor HMB-45, but was positive for Mi and S-100 (FIG. 7). Mi also stainedpositively in a melanoma-in-transit (FIG. 7). This invasive tumorresides in the dermal/subdermal region without contiguous extension fromoverlying epidermis.

Several specific clinical scenarios are shown in FIG. 8, which highlightinstances in which Mi can display particular diagnostic utility. AnHMB-45 negative melanoma is shown (FIG. 8, column 1) in which nuclearstaining for Mi is observed. Another tumor was HMB-45 positive only inthe in-situ component (FIG. 8, column 2, arrow), but HMB-45 negativewithin the invasive component (FIG. 8, column 2 labeled invasive). Incontrast, Mi staining was positive in both the in situ and invasivecomponents of this tumor (FIG. 8, column 2). One of the S-100 negativetumors is also shown (FIG. 8, column 3) which also was negative forHMB-45. It, too, stained positively for Mi. Finally, a melanoma is shownin which the Mi stain permitted detection of invasive tumor cellclusters deep within the sample, which might otherwise have been missed(FIG. 8, column 4, see arrow).

Rare variants of melanoma were evaluated for Mi staining. These variantsrepresent <1% of melanomas and were not seen in the consecutive seriesof 76 melanomas presented. Two pure spindle cell melanomas were stainedfor S-100, HMB-45, and Mi. One was positive for all three markers, andthe other was HMB-45 negative, but positive for Mi and S-100. Nine casesof desmoplastic/neurotropic melanoma were also stained with the 3markers. All were negative for both Mi and HMB-45, but positive forS-100. Thus while Mi was positive in 100% of melanomas in theconsecutive series, like HMB-45 it failed to detect 9 of 9desmoplastic/neurotropic melanomas.

To assess the specificity of Mi expression among non-melanoma tumors, 81non-melanocytic tumors were stained for Mi (Table 1). These samplescomprised 10 invasive ductal carcinomas of the breast, 10 squamous cellcarcinomas of the lung, 10 endometrial adenocarcinomas, 10 thyroidcarcinomas, 10 vulvar squamous cell carcinomas, 10 testicularcarcinomas, 4 schwannomas, 2 neurofibromas, 1 microcystic adnexalcarcinoma, and non-melanoma skin tumors (4 basal cell carcinomas, 4squamous cell carcinomas, 4 atypical fibroxanthomas, 2 granular celltumors). Of these tumors, all were negative for Mi nuclear staining, buttwo breast carcinomas displayed a cytoplasmic staining pattern. Only onethyroid carcinoma displayed focal HMB-45 staining, with the remainingcases being HMB-45 negative. Thus Mi stained cytoplasmic in 2 of 81non-melanomas, but no cases exhibited nuclear staining.

Table 1: Staining characteristics in 76 consecutive melanomas, rarevariants, non-melanoma tumors, and non-melanoma skin tumors. Number ofpositive cases is given in the numerator and total number of cases inthe denominator. Parentheses give percent positive cases. Asteriskindicates that 2 of the 10 breast cancer cases showed cytoplasmicstaining for Mi, but were scored negative due to lack of nuclearstaining.

TABLE 1 Mi+ HMB-45+ S-100+ (% positive) (% positive) (% positive)Consecutive series of Melanomas Melanoma in situ 19/19 (100) 19/19 (100)17/19 (89) Melanoma 50/50 (100) 44/50 (88) 48/50 (96) Amelanoticmelanoma 8/8 (100) 7/8 (88) 7/8 (88) Metastatic Melanoma 7/7 (100) 6/7(86) 6/7 (88) All melanomas in series 76/76 (100) 69/76 (91) 71/76 (93)Rare variants of Melanoma Desmoplastic/neurotropic 0/9 (0) 0/9 (0) 9/9(100) Pure spindle cell melanoma 2/2 (100) 1/2 (50) 2/2 (100)Non-Melanoma tumors Invasive Ductal 0/10* (0) 0/10 (0) Breast CarcinomaSquamous Carcinoma Lung 0/10 (0) 0/10 (0) Endometrial adenocarcinoma0/10 (0) 0/10 (0) Thyroid Carcinoma 0/10 (0) 1/10 (10) SquamousCarcinoma Vulva 0/10 (0) 0/10 (0) Testicular Cancer 0/10 (0) 0/10 (0)Schwannoma 0/4 (0) Microcystic Adenexal 0/1 (0) Carcinoma Neurofibroma0/2 (0) Non-melanoma Skin tumors Basal Cell Carcinoma 0/4 SquamousCarcinoma Skin 0/4 Atypical Fibroxanthomas 0/4 Granular Cell tumors 0/2All non-melanomas in series 0/81 (0) 1/60 (2)

In this series of 76 consecutively accessioned melanomas the Mi antibodydetected 100% of cases. S-100 and HMB-45 failed to detect 5 and 7 casesrespectively of the 76 melanomas. Mi also identified an area of deepdermal staining on a specimen that was difficult to visualize with H&Eand negative with HMB-45 staining. This deep staining may confer a worseprognosis because it would alter the measured thickness of the tumor andthereby alter treatment decisions such as optimal surgical marginsand/or adjuvant therapy. Mi is also a very specific antibody, stainingnuclei in none of 81 non-melanomas, though staining cytoplasms in 2. Inthis series, compared to current standard markers of melanoma, Mi ismore specific than S-100 and is as sensitive, if not more, than HMB-45.

REFERENCES

-   1. Silver, W. K. (1979) The coat colors of mice: a model for    mammalian gene action and interaction. (Spinger-Verlag,    Incorporated, New York).-   2. Hughes, A. E., Newton, V. E., Liu, X. Z. & Read, A. P. (1994) Nat    Genet 7, 509-12.-   3. Tassabehji, M., Newton, V. E. & Read, A. P. (1994) Nat. Genet. 8,    251-5.-   4. Hodgkinson, C. A., Moore, K. J., Nakayama, A., Steingrimsson, E.,    Copeland, N. G., Jenkins, N. A. & Arnheiter, H. (1993) Cell 74,    395-404.-   5. Hemesath, T. J., Steingrimsson, E., McGill, G., Hansen, M. J.,    Vaught, J., Hodgkinson, C. A., Arnheiter, H., Copeland, N. G.,    Jenkins, N. A. & Fisher, D. E. (1994) Genes Dev. 8, 2770-80.-   6. Yasumoto, K., Yokoyama, K., Shibata, K., Tomita, Y. &    Shibahara, S. (1994) Mol. Cell. Biol. 14, 8058-70.-   7. Bentley, N. J., Eisen, T. & Goding, C. R. (1994) Mol. Cell. Biol.    14, 7996-8006.-   8. Bertolotto, C., Abbe, P., Hemesath, T. J., Bille, K., Fisher, D.    E., Ortonne, J.-P. & Ballotti, R. (1998) J. Cell Biol. 142, 827-35.-   9. Price, E. R., Horstmann, M. A., Wells, A., Weilbacher, K. N.,    Takemoto, C. M., Landis, M. W. & Fisher, D. E. submitted.-   10. Lerner, A. B., Shiohara, T., Boissy, R. E., Jacobson, K. A.,    Lamoreux, M. L. & Moellmann, G. E. (1986) J. Invest. Dermatol. 87,    299-304.-   11. Steingrimsson, E., Moore, K. J., Lamoreux, M. L.,    Ferre, D. A. A. R., Burley, S. K., Zimring, D. C., Skow, L. C.,    Hodgkinson, C. A., Arnheiter, H., Copeland, N. G. & et al. (1994)    Nat. Genet. 8, 256-63.-   12. Hemesath, T. J., Price, E. R., Takemoto, C., Badalian, T. &    Fisher, D. E. (1998) Nature 391, 298-301.-   13. Price, E. R., Horstmann, M. A., Wells, A. G., Weilbacher, K. N.,    Takemoto, C. M., Landis, M. W. & Fisher, D. E. (1998) J Biol. Chem.    In Press.-   14. Sato, S., Roberts, K., Gambino, G., Cook, A., Kouzarides, T. &    Goding, C. R. (1997) Oncogene 14, 3083-92.-   15. Halaban, R., Bohm, M., Dotto, P., Moellmann, G., Cheng, E. &    Zhang, Y. (1996) J Invest Dermatol 106, 1266-72.-   16. Barth, A., Wanek, L. A. & Morton, D. L. (1995) J. Am. Coll.    Surg. 181, 193.-   17. Evans, G. R. D. & Manson, P. N. (1994) J. Am/Coll. Surg. 178,    523.-   18. DeVita, V., Hellman, S. & Rosenberg, S. (1993) Cancer:    Principles and Practice of Oncology (J. B. Lippincott Company,    Philadelphia).-   19. Chang, P. & Knapper, W. (1982) Cancer 49, 1106-1111.-   20. Jonk, A., Kroon, B., Rumke, P., Mooi, W., Hart, A. & Van    Dongen, J. (1990) Br J Surg 77, 665-8.-   21. Reintgen, D., McCarty, K., Woodard, B., Cox, E. &    Seigler, H. (1983) Surg, Gynecol&Obstet 156, 335-40.-   22. Schlagenhauff, B., Stroebel, W., Ellwanger, U., Meier, F.,    Zimmermann, C., Breuninger, C., Rassner, G. & Garbe, C. (1997)    Cancer 80, 60-5.-   23. Kaufmann, O., Koch, S., Burghard, J., Audring, H. &    Dietel, M. (1998) Mod Pathol 11, 740-746.-   24. Zakut, R., Perlis, R., Eliyahu, S., Yarden, Y., Givol, D.,    Lyman, S. & Halaban, R. (1993) Oncogene 8, 2221-2229.-   25. Halaban, R., Cheng, E., Zhang, Y., Moellmann, G., Hanlon, D.,    Michalak, M., Setaluri, V. & Hebert, D. N. (1997) Proc Natl Acad Sci    USA 94, 6210-5.-   26. Montone K. T., van Belle, P. Elenitsas R., & Elder, D. E. (1997)    Mod Pathol 10(9):939-44.-   27. Rossi, C., Foletto, A., Vecchiato, S., Alessio, N., Menin, N. &    Lise, M. (1997) Eur J Cancer 33, 2302-12.-   28. Cochran, A. & Wen, D. (1985) Pathology 17, 340-345.-   29. Orchard, G. & Jones, E. (1994) Britis Journal of Biomedical    Science 51, 44-56.-   30. Bacchi, C., Bonetti, F., Pea, M., Martignoni, G. &    Gown, A. (1996) Applied Immunhistochemistry 4, 73-85.-   31. Kahn, H., Marks, A., Thom, H. & Baumal, R. (1983) Am J Clin    Pathol 79, 341-347.-   32. Busam, K., Chen, Y., Old, L., Stockert, E., Iversen, K., Coplan,    K., Rosai, J., Barnhill, R. & Jungbluth, A. (1998) Am J Surg Pathol    22, 976-982.-   33. Elenitsas, R. & Schuchter, L. (1998) Current opinion in oncology    10, 162-169.-   34. Skelton, H., Maceira, J., Smith, K., McCarthy, W., Lupton, G. &    Graham, J. (1997) Am J Dermatopathol 19, 580-4.-   35. Gown, A., Vogel, A., Hoak, A., Gough, F. & NcNutt, M. (1986) Am    J Pathol 123, 195-203.-   36. Cohen, T., Gitay-Gorey, H., Sharon, R., Shibuya, M., Halaban,    R., Levi, B. & Neufeld, G. (1995) J Biol Chem 270, 1132-11326.-   37. Weilbaecher, K. N., Hershey, C. L., Takemoto, C. M.,    Horstmann, M. A., Hemesath, T. J., Tashjian, A. H. &    Fisher, D. E. (1998) J Exp Med 187, 775-85.-   38. Ackerman, L. (1953) Surgical Pathology (Mosby, St. Louis).-   39. Schmitt, F. & Bachhi, C. (1989) Histopathology 15, 281-8.-   40. Stefansson, K., Wollmann, R. & Jerkovic, M. (1982) Am J Pathol    106, 261-8.-   41. Guillermo, A., Herrera, E., Turbat-Herrera, A. & al., e. (1988)    Am J Clin Pathol 89, 168-76.-   42. Drier, J., Swanson, P., Cherwitz, D. & Wick, M. (1987) Arch    Pathol Lab Med 111, 447-52.-   43. Zarbo, R., Gown, A., Visscher, D. & Crissman, J. (1990) Mod.    Pathol. 3, 494-501.-   44. Yoneda, T., Sasaki, A. & Mundy, G. (1994) Breast Cancer Research    and Treatment 32, 73-84.-   45. Scher, H. & Yagoda, A. (1987) American J Medicine 82, 6-28.-   46. Littlewood-Evans, A., Bilbe, G., Bowler, W., Farley, D.,    Wlordski, B., Kokubo, T., Inaoka, T., Sloane, J., Evans, D. &    Gallager, J. (1997) Cancer Research 57, 5386-90.-   47. Carlson, J., Dickerson, G. & Sober, H. (1995) Cancer 75, 478.-   48. Labrecque, P., Hu, C. & Winkelmann, R. (1976) Cancer 38,    1205-13.-   49. DiMaio, S. M., Mackay, B., Smith, J. L. J. &    Dickersin, G. R. (1982) Cancer 50, 2345-54.-   50. Weiss & Enzinger (1983), pp. 919-921.

The references described herein are incorporated herein by reference.

1.-4. (canceled)
 5. A method for determining whether a malignant cell isa melanoma comprising: determining whether melanoma microphthalmia (Mi)is being expressed in a nucleus of the malignant cell by using a probefor melanoma Mi, wherein the expression of melanoma Mi is indicative ofthe malignant cell being a melanoma.
 6. The method of claim 5, whereinthe probe is an antibody for melanoma Mi.
 7. The method of claim 6,wherein the antibody is a monoclonal antibody. 8.-10. (canceled)
 11. Themethod of claim 5, wherein the level of melanoma Mi present in thenucleus is measured and compared to a base line control level ofmelanoma Mi.
 12. The method of claim 5, wherein the activation state ofthe melanoma Mi in the nucleus is determined.
 13. The method of claim 5,wherein the probe is a nucleic acid.
 14. The method of claim 13, whereinthe nucleic acid is DNA.
 15. The method of claim 13, wherein the nucleicacid is mRNA.